Varicella-zoster virus (VZV) is a human-restricted alphaherpesvirus. It causes varicella (chicken pox) upon primary infection and zoster (shingles) upon reactivation from latency. VZV disease is partially preventable by inoculation with the live, attenuated vaccine strain Oka-Merck (Oxman et al., 2005; Vazquez et al., 2004). Pediatric vaccination has reduced varicella cases in the United States (Seward et al., 2008), although the incidence of zoster is not likely to decline in the near future because in older adults the vaccine efficacy is approximately 50% (Holcomb and Weinberg, 2006). There will continue to be a demand for antiviral drugs for VZV due to natural and breakthrough cases and in immunocompromised patients that cannot receive live virus vaccines. Current treatments are nucleoside and pyrophosphate analogues that target the virus DNA polymerase and may depend on virus thymidine kinase activity (De Clercq, 2004). Acyclovir (ACV) and its derivatives valaciclovir (VACV), penciclovir (PCV) and famciclovir (FCV) are acyclic derivatives of guanine. They are moderately effective against VZV, but for best results treatment should begin within 72 h of rash onset and resistance may arise during long-term administration to immunocompromised patients (Sampathkumar et al., 2009). In these patients, Foscarnet (phosphonoformate) delivered intravenously may be necessary to treat resistant VZV (Ahmed et al., 2007). These drugs are widely approved for use in the United States, Europe, and Asia.
The cyclic derivatives of uridine are another class of drugs currently used to treat VZV. Infections in the eye (herpes zoster ophthalmicus) can be treated with topical idoxuridine and trifluridine. Brivudin [BVDU, (E)-5-(2-bromovinyl)-2′-deoxyuridine] is approved for use in Europe and was the first bromovinyl nucleoside analog to show anti-herpesvirus activity (De Clercq et al., 1979). BVDU is phosphorylated by the virus-encoded thymidine kinase (TK) to both the 5′-monophosphate and 5′-diphosphate forms. Cellular kinases produce the 5′-triphosphate form (BVDU-TP). BVDU-TP interacts with the viral DNA polymerase either as a competitive inhibitor or an alternative substrate whereby it can be incorporated into the DNA chain (reviewed in (De Clercq, 2005)). BVDU is more potent against VZV than acyclovir and its derivatives (Andrei et al., 1995; Shigeta et al., 1983). Another benefit of BVDU over acyclovir is the ease of dosing, making it appealing to elderly patients (De Clercq, 2005). The main drawback of BVDU is that it is cleaved into a metabolite of BVU. BVU in turn inhibits dihydropyrimidine dehydrogenase, which is involved in the degradation of thymidine, uracil, and the commonly used cancer drug 5′-fluorouracil (5-FU). Patients receiving this chemotherapy regimen should not be given BVDU as it may cause toxic accumulation of 5-FU and result in death [reviewed in (De Clercq, 2004; De Clercq, 2005; Diasio, 1998; Keizer et al., 1994)].
The serious possible adverse effects of BVDU are the main reason why related compounds have been screened for antiviral activity without the potential toxicity. One approach has been to screen nucleosides in the non-naturally occurring L-configuration, which can be just as effective as the D-nucleoside counterparts (Chu et al., 1995; Spadari et al., 1992). The uridine derivative, β-L-1-[5-(E-2-Bromovinyl)-2-(hydroxymethyl)-1,3-dioxolan-4-yl)]uracil (L-BHDU), exhibited potent anti-VZV activity in cultured cells and it was noncytotoxic in HEL 299 cells up to 200 μM (Choi et al., 2000; Li et al., 2000). Efforts to elucidate the mechanism of action found that L-BHDU was phosphorylated by VZV TK but not further converted to the di- and triphosphate forms. This is different from BVDU and implies an alternative antiviral mechanism (Li et al., 2000). Their evidence pointed to the monophosphate form as the active moiety that would inhibit VZV DNA polymerase. The next question regarding this promising compound was whether it was effective against VZV in vivo.
In this study, we evaluated L-BHDU in a range of models that address cytotoxicity and efficacy in culture and in vivo. We have developed systems for screening potential antiviral compounds against VZV that employ fully differentiated, intact human tissues and live animals in an attempt to more closely mimic what occurs during a natural infection (Rowe et al.). The cytotoxic and antiviral effects of L-BHDU were first examined in a primary cell line, human foreskin fibroblasts (HFFs), and then ex vivo in a skin organ culture (SOC) model (Taylor and Moffat, 2005). Finally, the effects of L-BHDU were tested against VZV in SCID-Hu mice with human skin xenografts (Moffat and Arvin, 1999). This screening process employs the recombinant strain VZV-BAC-Luc, which was selected for its expression of firefly luciferase that can be quantitatively measured by bioluminescence, as well as for its wild type virulence and tissue tropism (Zhang et al., 2007). We report that L-BHDU prevented VZV replication in HFFs as wells as in skin explants and xenografts in the SCID-Hu mouse. This demonstrates the potential of L-BHDU as a novel anti-VZV agent.